Directed evolution is a process wherein the sequence of a gene is varied randomly by any of a number of methods, generating a library of mutated genes. These mutated genes are expressed and the functions of those gene products are assayed. A selection procedure is then applied to select those cells containing genes that express products-with desirable functions. These cells, and their genes, are then selectively amplified, and the mutagenesis, screening and selection process is repeated until gene products with the most desirable functions are obtained.
The general scheme for directed evolution is shown in FIG. 1. First, variation is introduced into the gene in question by some type of random mutagenesis and a library of sequences is introduced into an organism (typically Escherichia coli) for expression of the altered proteins. Next, this population of bacteria is screened for the desired activity and individual colonies are selected. Finally, these selected bacteria are grown up (amplification of the selected genetic variants) and the plasmids expressing proteins with the most desirable functional traits are isolated. These then are used as heterogeneous templates for further random mutagenesis and reintroduced into the bacterium for another round of screening and amplification. This cycle is continued until the desired functional characteristics are achieved.
Directed evolution has been successfully used to generate new molecules with altered physical characteristics. For example, Doi et al. modified green fluorescent protein (GFP) to include a binding site for the TEM1-lactamase inhibitor and then used directed evolution methods to produce a protein molecule whose fluorescent properties changed upon binding the target molecule. N. Doi and H. Yanagawa (1999) “Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution,” FEBS Letters 453, 305–307). Directed evolution methodologies involving fluorescent proteins are particularly useful as fluorescence lends itself to sensitive and relatively easy, albeit slow, visual measurement.
GFP is one of a few different proteins that, in the absence of any externally supplied cofactor, fluoresce strongly in the visible region of the spectrum. Two of these proteins, GFP and a related red fluorescing protein (RFP) from reef corals, are commercially available in the form of expressible plasmids. Tsien, R. Y. (1998) “The Green Fluorescent Protein,” Annu. Rev. Biochem. 67:509–544; Matz, V., et al. (1999) “Fluorescent proteins from nonbioluminescent Anthozoa species,” Nature Biotechnology 17: 1969–1973. Functional transgenic expression of these fluorescent proteins is nearly universal in both eukaryotes and prokaryotes. Both the green and red fluorescing proteins have similar structural features, involving a beta-can fold structure enclosing a chromophore that is made via a reaction between 3 consecutive amino acids, serine, tyrosine and glycine. The quantum yield of fluorescence from the green fluorescent protein is near unity, while that from the red protein is apparently lower. Proteins with a variety of intermediate wavelengths have also been characterized.
Most of the directed evolution studies performed to date have utilized visual, qualitative screening of colonies on plates followed by manual selection of colonies that have enhanced activity in the protein of interest. Selection of cells may be based on a number of criteria, including color, morphology, size and fluorescence, depending on the protein of interest and the selectable marker chosen. When screening fluorescing cells, the process typically involves exciting cells with light and observing fluorescence from the genes or from molecules made by or associated with the genes in the cells. This visual screening process is slow and not particularly amenable to automation. As a result, the number of cells that can be screened and selected for further processing is greatly limited.
Although electronic cameras have been used to record fluorescence levels from colonies of cells, only the total relative yield of the fluorescence is recorded. This does not distinguish between fluorescence amplitude, which depends on both the photophysical properties of the fluorophore and its concentration, and fluorescence lifetime, which depends only on the photophysical properties of the fluorophore. Thus, directed evolution procedures that rely on steady state measurements of fluorescence select for changes that can be in either the amount of or the chemical properties of the fluorophore, but cannot specifically select for changes in molecular properties independent of concentration.
Also, while the use of electronic cameras has made it possible to screen cells more rapidly, its application has been limited by the ability to manually select cells exhibiting desired traits. What is needed, therefore, is a more sensitive, higher resolution system that quantitates levels of fluorescence from microcolonies (colonies with a diameter of approximately 100 microns or less) or from individual cells, thus allowing cell screening on the order of millions of cells per round of directed evolution, coupled with an automated system for selecting the microcolonies or cells of interest.
Thus, the ability to perform directed evolution using a high resolution fluorescent assay that is sensitive, amenable to automation, and that distinguishes between fluorescence amplitude and fluorescence lifetime would be a significant asset for research as well as diagnostics and therapeutics.